The lymph node serves as an in-line filter of the lymphatic and interstitial circulation, containing fluids, particulates and cells, in particular dendritic cells (DCs), while also receiving leukocytes from the circulatory system, ultimately for the purpose of systemic immune surveillance against infectious agents and malignancy. Given these key physiological functions, we deem it important to develop a three-dimensional (3D) biomolecular tissue map of the normal human lymph node (Organ Specific Project 3 [OSP 3]). With over a decade of experience in the procurement of transplant-quality organs for research, including those of the lymphatic system, approximately 600 human lymph nodes banked in our biorepository, and a robust research pipeline, we are well equipped to evaluate in 3D space, specific cellular interactions, gene and protein expression patterns, as well as protein modifications that govern regional immune activation, anergy, and tolerance activities in this secondary lymphoid organ. To achieve this goal, we intend to perform systematic, anatomic dissection of freshly acquired human lymph nodes, with assessment of normality by an independent pathologist, to support all subsequent AIMs (AIM I). We will generate macro images of the intact organ and lymph node sections (AIM IIA) to guide microanatomic analyses. Our microscopic imaging platforms (AIM IIB-D) include immunohistochemistry (IHC) and immunofluorescence (IF) with widefield, confocal, multiphoton, and Stochastic Optical Reconstruction Microscopy (STORM) imaging, along with optical clearance with light sheet fluorescence microscopy (LSFM) and Pathology-optimized Expansion Microscopy (ExPATH). These applications will then be coupled with histology topography cytometry analysis toolbox (histoCAT), FluoRender and Amira-Avizo Software to enable 3D reconstruction (Data Core). Dispersed lymph node cells will be immunophenotyped and isolated by fluorescence activated cell sorting (FACS) to characterize the normal T cell receptor (TCR) and B cell receptor (BCR) repertoire (AIM III). Biomolecular analysis of tissue sections by imaging mass cytometry (IMC) (AIM IVA) offers the ability to simultaneously visualize dozens of proteins, their modifications, and RNA species at the cellular level with 3D reconstruction. This, coupled with single molecule fluorescence in situ hybridization (smFISH) analyses and single cell RNA-Seq using 10x GENOMICS GemCode and 10x Chromium Technology (AIM IVB) could facilitate discovery of novel cellular markers relevant to human lymphatic anatomy and immunological function. These novel markers will then after validation be applied to the optical and IMC platforms for 3D location within the lymph node. Both raw and analyzed data generated at in each stage of the research pipeline will be integrated into the HIVE via interactions with the Data Core, the Coordination Core, and other TMC efforts. We believe these proposed studies represent a critical step toward a highly multiplexed, high- resolution analysis of the lymph node macro- and microanatomy to serve as a catalyst for subsequent studies in disease-specific arenas.